Reconstitution of lethally irradiated mice with a mixture of T cell depleted syngeneic and allogeneic bone marrow leads to the induction of stable mixed lymphohematopoietic chimerism, full immunocompetence, and specific transplantation tolerance. However, the potential clinical applicability of this approach is severely limited by the toxicity of lethal irradiation as a preparative regimen. We have recently described a non-myeloablative preparative regimen likewise capable of inducing stable mixed lymphohematopoietic chimerism, in which anti-CD4 and anti- CD8 monoclonal antibodies are used to eliminate mature T cells from the host. Following this mAb treatment, relatively non-toxic whole body irradiation at 3 Gy plus 7 Gy thymic irradiation were sufficient to permit engraftment of fully MHC mismatched allogeneic bone marrow. The major goals of this proposal are to examine the influence of various genetic barriers on the induction of mixed lymphohematopoietic chimerism in this system and to determine the mechanism of the tolerance induced. Specifically, we intend: 1) To evaluate the role of T cell subsets and NK cells in resisting engraftment across selected MHC and non-MHC barriers; 2) To determine the mechanism whereby maintenance of tolerance depends on the persistence of allogeneic lymphohematopoietic elements; 3) To determine the ability of tolerance to withstand challenge with normal nontolerant recipient-strain T cells; 4) To evaluate the susceptibility of chimeras prepared by the non-myeloablative conditioning regimen to the induction of GVHD by administration of donor T cells either at the time of BMT or following the establishment of stable mixed chimerism; and 5) To evaluate clonal deletion of specific alloreactive T cells by in vitro assays of cellular immunity and by detection of recipient T cells bearing donor-reactive Vbeta by flow cytometry. These studies should provide insight into the mechanism of the tolerance induced by this new preparative regimen as well as into the potential applicability of this methodology for induction of specific transplantation tolerance across different histocompatibility barriers.